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US 7,252,940 B2Granted
Abasic site endonuclease assay
AssigneeEpoch Biosciences, Inc.
InventorsIgor V. Kutyavin, David Milesi, Merl F. Hoekstra
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Abstract
The present invention provides a novel method for detection and/or genotyping of nucleic acids that utilizes the specificity of an AP endonuclease. In addition, the present invention provides a novel method for nucleic acid amplification.
Claims28
A method of detecting a target nucleic acid in a sample, comprising:
- a) contacting the sample with at least one AP site probe and an AP endonuclease, under conditions sufficient to allow the AP site probe to hybridize to the target nucleic acid and form a reaction mixture, wherein said AP site probe comprises an oligonucleotide NA that hybridizes to the target nucleic acid and a functional tail R comprising a detectable reporter group, said functional tail R attached via a phosphodiester bond of a phosphate group to the 3′ terminal nucleotide of the NA, wherein the reporter group is not detected when the functional tail R is attached to the NA;
- and
- b) incubating the reaction mixture under reaction conditions sufficient to allow said AP endonuclease to cleave the phosphodiester bond attaching the functional tail R to the 3′ terminal of the NA, wherein the AP endonuclease preferentially cleaves the phosphodiester bond attaching the tail R to the NA when the NA is hybridized with a complementary target nucleic acid sequence in comparison to when the NA is unhybridized or hybridized to a non-complementary target nucleic acid;
- and
- c) detecting the reporter group on the cleaved functional tail R, whereby the target nucleic acid is detected.
2 more elements
A method of detecting a target nucleic acid in a sample, comprising:
- a) contacting the sample with at least one AP site probe and an AP endonuclease, under conditions sufficient to allow the AP site probe to hybridize to the target nucleic acid and form a reaction mixture, wherein said AP site probe comprises an oligonucleotide NA that hybridizes to the target nucleic acid, a functional tail R comprising a quencher molecule, said functional tail R attached via a phosphodiester bond of a phosphate group to the 3′ terminal of the NA, and a reporter group attached via a non-cleavable linker to the 5′ terminal of the NA, wherein the reporter group is not detected when the functional tail R is attached to the NA;
- and
- b) incubating the reaction mixture under reaction conditions sufficient to allow said AP endonuclease to cleave the phosphodiester bond attaching the functional tail R to the 3′ terminal of the NA, wherein the AP endonuclease preferentially cleaves the phosphodiester bond attaching the tail R to the NA when the NA is hybridized with a complementary target nucleic acid sequence in comparison to when the NA is unhybridized or hybridized to a non-complementary target nucleic acid;
- and
- c) detecting the reporter group upon cleavage of the functional tail R, whereby the target nucleic acid is detected.
2 more elements
Dependencies are inferred from the claim text.